#METABOLOMICS WORKBENCH pttnguyen27_20241215_201027 DATATRACK_ID:5454 STUDY_ID:ST003644 ANALYSIS_ID:AN005983 PROJECT_ID:PR002254 VERSION 1 CREATED_ON December 31, 2024, 5:42 am #PROJECT PR:PROJECT_TITLE Acetyl-CoA synthesis in the skin is a key determinant of systemic lipid PR:PROJECT_TITLE homeostasis PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY ATP-citrate lyase (ACLY) generates cytosolic acetyl-CoA for lipid synthesis and PR:PROJECT_SUMMARY is a promising therapeutic target in diseases with altered lipid metabolism. PR:PROJECT_SUMMARY Here, we developed inducible whole-body Acly knockout mice to determine the PR:PROJECT_SUMMARY requirement for ACLY in normal tissue functions, uncovering its crucial role in PR:PROJECT_SUMMARY skin homeostasis. ACLY-deficient skin upregulates the acetyl-CoA synthetase PR:PROJECT_SUMMARY ACSS2; deletion of both Acly and Acss2 from the skin exacerbates skin PR:PROJECT_SUMMARY abnormalities, with differential effects on two major lipid-producing skin PR:PROJECT_SUMMARY compartments. While the epidermis is depleted of barrier lipids, the sebaceous PR:PROJECT_SUMMARY glands increase production of sebum, supplied at least in part by circulating PR:PROJECT_SUMMARY fatty acids and coinciding with adipose lipolysis and fat depletion. Dietary fat PR:PROJECT_SUMMARY supplementation further boosts sebum production and partially rescues both the PR:PROJECT_SUMMARY lipoatrophy and aberrant skin phenotypes. The data establish a critical role for PR:PROJECT_SUMMARY cytosolic acetyl-CoA synthesis in maintaining skin barrier integrity and PR:PROJECT_SUMMARY highlight the skin as a key organ in systemic lipid regulation. PR:INSTITUTE University of Pennsylvania PR:DEPARTMENT Cancer Biology PR:LABORATORY Wellen PR:LAST_NAME Nguyen PR:FIRST_NAME Phuong PR:ADDRESS 421 Curie Boulevard, Philadelphia, Pennsylvania, 19104, USA PR:EMAIL pttnguyen27@gmail.com PR:PHONE 2674376821 PR:PUBLICATIONS Nguyen, P. et al Acetyl-CoA synthesis in the skin is a key determinant of PR:PUBLICATIONS systemic lipid homeostasis #STUDY ST:STUDY_TITLE Mouse epidermal lipidomics ST:STUDY_SUMMARY Skin-specific double knockout (DKO) of acetyl-CoA synthesizing enzymes, ACLY and ST:STUDY_SUMMARY ACSS2, induces skin barrier dysfunction and systemic fat loss in mice that can ST:STUDY_SUMMARY be partially rescued by olive oil supplementation. In this study, lipidomics was ST:STUDY_SUMMARY performed for epidermis derived from DKO versus wild-type mouse skin with or ST:STUDY_SUMMARY without olive oil supplementation to examine the changes in epidermal lipid ST:STUDY_SUMMARY profile in response to limited acetyl-CoA availability and to dietary lipid ST:STUDY_SUMMARY intervention. ST:INSTITUTE University of Pennsylvania ST:DEPARTMENT Cancer Biology ST:LABORATORY Wellen ST:LAST_NAME Nguyen ST:FIRST_NAME Phuong ST:ADDRESS 421 Curie Boulevard, Philadelphia PA 19104 USA ST:EMAIL pttnguyen27@gmail.com ST:PHONE 2674376821 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 10 weeks SU:GENDER Male and female SU:ANIMAL_LIGHT_CYCLE 7am-7pm SU:ANIMAL_FEED Standard chow diet with or without olive oil gavage #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - BE-01 Sample source:extraction blank for exclusion list in MS | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_BE-01.mzML SUBJECT_SAMPLE_FACTORS - BE-01-inj-02 Sample source:extraction blank for exclusion list in MS | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_BE-01-inj-02.mzML SUBJECT_SAMPLE_FACTORS - BE-02 Sample source:extraction blank for exclusion list in MS | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_BE-02.mzML SUBJECT_SAMPLE_FACTORS - BE-02-inj-02 Sample source:extraction blank for exclusion list in MS | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_BE-02-inj-02.mzML SUBJECT_SAMPLE_FACTORS - ISTD-Ext-01 Sample source:internal standard extracted from water | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_ISTD-Ext-01.mzML SUBJECT_SAMPLE_FACTORS - ISTD-Ext-02 Sample source:internal standard extracted from water | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_ISTD-Ext-02.mzML SUBJECT_SAMPLE_FACTORS - ISTD-D-01 Sample source:internal standard dry | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_ISTD-D-01.mzML SUBJECT_SAMPLE_FACTORS - ISTD-D-02 Sample source:internal standard dry | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_ISTD-D-02.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-01-01 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-01-01.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-01-02 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-01-02.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-02-01 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-02-01.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-02-02 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-02-02.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-03-01 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-03-01.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-03-02 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-03-02.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-04-01 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-04-01.mzML SUBJECT_SAMPLE_FACTORS - QC_Pool-04-02 Sample source:Pooled from all 24 samples | Genotype:NA | Olive oil gavage:NA Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0329_QC_Pool-04-02.mzML SUBJECT_SAMPLE_FACTORS - D359 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-01.mzML SUBJECT_SAMPLE_FACTORS - D360 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-02.mzML SUBJECT_SAMPLE_FACTORS - D361 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-03.mzML SUBJECT_SAMPLE_FACTORS - D362 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-04.mzML SUBJECT_SAMPLE_FACTORS - D364 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-05.mzML SUBJECT_SAMPLE_FACTORS - D365 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-06.mzML SUBJECT_SAMPLE_FACTORS - D371 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-07.mzML SUBJECT_SAMPLE_FACTORS - D265 Sample source:Epidermis | Genotype:WT | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-WT-08.mzML SUBJECT_SAMPLE_FACTORS - D266 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-09.mzML SUBJECT_SAMPLE_FACTORS - D267 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-10.mzML SUBJECT_SAMPLE_FACTORS - D269 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-11.mzML SUBJECT_SAMPLE_FACTORS - D270 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-12.mzML SUBJECT_SAMPLE_FACTORS - D271 Sample source:Epidermis | Genotype:WT | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-WT-13.mzML SUBJECT_SAMPLE_FACTORS - D272 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-14.mzML SUBJECT_SAMPLE_FACTORS - D273 Sample source:Epidermis | Genotype:WT | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-WT-15.mzML SUBJECT_SAMPLE_FACTORS - D274 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-16.mzML SUBJECT_SAMPLE_FACTORS - D275 Sample source:Epidermis | Genotype:WT | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-WT-17.mzML SUBJECT_SAMPLE_FACTORS - D276 Sample source:Epidermis | Genotype:KO | Olive oil gavage:No Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-KO-18.mzML SUBJECT_SAMPLE_FACTORS - D284 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-19.mzML SUBJECT_SAMPLE_FACTORS - D285 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-20.mzML SUBJECT_SAMPLE_FACTORS - D286 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-21.mzML SUBJECT_SAMPLE_FACTORS - D287 Sample source:Epidermis | Genotype:KO | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-KO-22.mzML SUBJECT_SAMPLE_FACTORS - D288 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-23.mzML SUBJECT_SAMPLE_FACTORS - D289 Sample source:Epidermis | Genotype:WT | Olive oil gavage:Yes Mass spec polarity=positive; Batch=B1; RAW_FILE_NAME(File Name)=0328_Epi-Oil-WT-24.mzML #COLLECTION CO:COLLECTION_SUMMARY Epidermis was isolated from mouse skin with dispase enzyme incubation and flash CO:COLLECTION_SUMMARY frozen until processed for lipid extraction CO:SAMPLE_TYPE Epidermis CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Skin-specific double knockout (DKO) of ACLY and ACSS2 was induced by Tamoxifen TR:TREATMENT_SUMMARY injections (first injection was day 0). For the treated mice, olive oil gavage TR:TREATMENT_SUMMARY was given at 200uL per day on every other day starting at day 11 and ending at TR:TREATMENT_SUMMARY day 19. Epidermal tissue was collected on day 21. TR:TREATMENT_COMPOUND Olive oil TR:TREATMENT_ROUTE Gavage TR:TREATMENT_DOSE 200uL #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 5 mg of frozen epidermis was homogenized in 0.6 mL of ice-cold 80% methanol SP:SAMPLEPREP_SUMMARY using green bullet blender tubes (Next Advance NA-GREENR1-RNA) and the Next SP:SAMPLEPREP_SUMMARY Advance Bullet Blender Tissue Homogenizer with dry ice at max speed x3 for 5 SP:SAMPLEPREP_SUMMARY minutes each. The tubes were vortexed and allowed to settle and come to room SP:SAMPLEPREP_SUMMARY temp for 30 minutes. 125 µL of homogenate (equivalent to 1 mg of tissue) was SP:SAMPLEPREP_SUMMARY transferred into a 10 mL Pyrex Glass tube, followed by adding 20 µL of internal SP:SAMPLEPREP_SUMMARY standard mix (1:1, SPLASH® LIPIDOMIX mass spec standard #330707: SP:SAMPLEPREP_SUMMARY Ceramide/Sphingoid mixture I #LM6002, both from Avanti Polar Lipids), 80% SP:SAMPLEPREP_SUMMARY methanol up to 2 mL of total methanol, and 1.7 mL of chloroform. The mixture was SP:SAMPLEPREP_SUMMARY then shaken vigorously for 20 minutes at room temperature. Each sample was added SP:SAMPLEPREP_SUMMARY with 1.4 mL of deionized water, vortexed for 30 seconds, and centrifuged at 2000 SP:SAMPLEPREP_SUMMARY rpm for 10 minutes for phase separation. The bottom chloroform layer was SP:SAMPLEPREP_SUMMARY collected and dried down under nitrogen gas. Then, dried lipids were resuspended SP:SAMPLEPREP_SUMMARY in 200 µL of methyl tert-butyl ether:methanol (1:3, v/v), sonicated for 5 SP:SAMPLEPREP_SUMMARY minutes in water bath at room temperature, and centrifuged at 10,000 g for 10 SP:SAMPLEPREP_SUMMARY minutes at 4°C. A pooled sample was made by mixing 40 µL from each SP:SAMPLEPREP_SUMMARY re-dissolved sample. This pooled sample was used as quality control (QC) for the SP:SAMPLEPREP_SUMMARY LC-HRMS response and ran every 8 samples. The QC was used for data SP:SAMPLEPREP_SUMMARY normalization. The rest of the lipid re-dissolved sample was transferred to a SP:SAMPLEPREP_SUMMARY HPLC vial and 2 µL injections were made in both positive mode and separately in SP:SAMPLEPREP_SUMMARY the negative mode. Control extraction blanks were made using only solvents SP:SAMPLEPREP_SUMMARY instead of the tissue homogenate. The control extraction blanks were used for SP:SAMPLEPREP_SUMMARY the exclusion list with a threshold feature intensity set at 1e105. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Ultimate 3000 UPLC system CH:COLUMN_NAME Thermo Accucore C18 (150 x 2.1mm,2.6um) CH:SOLVENT_A 50% Acetonitrile/50% Water; 0.1% formic acid; 10 mM ammonium formate CH:SOLVENT_B 10% Acetonitrile/88% Isopropanol/2% Water; 0.02% formic acid; 2 mM ammonium CH:SOLVENT_B formate CH:FLOW_GRADIENT 0 min, 90% A; 1 min, 90% A; 4 min, 60% A; 12 min, 25% A; 21 min, 1% A; 24 min, CH:FLOW_GRADIENT 1% A; 24.1 min, 90% A; and 28 min, 90% A CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 35℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS All samples were analyzed in a randomized order in full scan MS (for MS:MS_COMMENTS quantification) that alternated with MS2 (for identification) of top 10, with MS:MS_COMMENTS HCD scans at 30, 45 or 60 eV. Data acquisition was performed in Full Scan/ddMS2 MS:MS_COMMENTS mode @ 120,000 resolution in positive mode. The Full Scan settings were as MS:MS_COMMENTS follows: AGC target, 1e6; Maximum IT, 250 ms; and scan range, 250–1800 m/z. MS:MS_RESULTS_FILE ST003644_AN005983_Results.txt UNITS:Area under the curve Has m/z:Yes Has RT:Yes RT units:Minutes #END